When plural random amino acid sequences are introduced into one site of a protein, oligonucleotides synthesized at random are introduced into the gene of the protein. However, when an introduced sequence is synthesized completely at random (NNN sequence), a stop codon emerges at a ratio of about 1/21. In addition, when synthesized an oligonucleotide to be introduced is a long strand, an oligonucleotide having deleted or inserted bases is found in a proportion of a few percent. Since they markedly reduce the diversity of the resulting protein library, those oligonucleotides need to be removed during preparation of the oligonucleotide library.
As a method for removing such unnecessary oligonucleotides from a synthesized oligonucleotide library, a method utilizing antibiotic drug selection can be mentioned. For example, a random oligonucleotide library to be the maturation target is ligated to the 5′ terminus of a drug resistance gene such as β-lactamase, and introduced into a plasmid. Escherichia coli is transformed with this plasmid and applied to an agar plate containing the corresponding antibiotic. As a result, only Escherichia coli having an in-frame gene without a stop codon or a frame shift due to deletion or insertion of bases can grow as a colony on the plate, since such Escherichia coli can express a drug resistance protein. Therefore, by recovering a gene from such colonies, a maturated oligonucleotide library free of unnecessary oligonucleotides can be obtained.
However, this method has some problems. Firstly, it is difficult to form a large number of colonies by a general method, since the number of colonies of Escherichia coli varies depending on the ligation efficiency of oligonucleotide library to a vector and transformation efficiency of Escherichia coli. Secondly, to obtain a large number of colonies, the corresponding number of samples needs to be prepared, which requires a lot of efforts and time.
The period that the present inventors required to maturate an oligonucleotide library having 108 diversities by this method was about 1 week. When an oligonucleotide library encoding an antibody gene library wherein 6 CDRs (complementary-determining regions) of the antibody are randomized is prepared, oligonucleotide libraries encoding each of these 6 CDRs are sequentially maturated, and maturation of the whole oligonucleotide library encoding the antibody gene library requires about 6 weeks.
Furthermore, since the above-mentioned manipulation requires large amounts of expensive reagents (enzymes etc.), the method is extremely costly.
On the other hand, patent document 1 discloses a ribosome display. In addition, patent document 1, paragraph [0051], discloses mRNA having a sequence encoding a translation reaction elongation arrest sequence of Escherichia coli SecM at the downstream of a spacer sequence.